Modification of the triplet repeat primed polymerase chain reaction method for detection of the CTG repeat expansion in myotonic dystrophy type 1: application in preimplantation genetic diagnosis

Kakourou, Georgia; Dhanjal, Seema; Mamas, T; Serhal, Paul; Delhanty, Joy D. A.; SenGupta, Sioban

doi:10.1016/j.fertnstert.2009.10.050

Cited by 18 publications

(20 citation statements)

References 27 publications

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“…Reaction mix contained PCR grade water, 1x Expand Long Template Buffer 3 (with 27.5 mM MgCl 2 and detergents), dNTPs (350 mM each), 2 mM each primer, 1.5 units Expand Long Template enzyme mix and 1 mL of SurePlex amplified product for a final volume of 15 mL. Thermal cycling was carried out for 45 cycles as described by Kakourou et al 2010 (annealing temperature was 54.5 C). After amplification of the sequences involving the two mutations, the amplified products were cleaned using EXOSAP-IT (Affymetrix, UK) and minisequencing was carried out using the SNaPshot Multiplex kit (Applied Biosystems, UK) according to manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

Successful Live Birth following Preimplantation Genetic Diagnosis for Phenylketonuria in Day 3 Embryos by Specific Mutation Analysis and Elective Single Embryo Transfer

et al. 2012

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Phenylketonuria (PKU) is an autosomal recessive inherited metabolic disorder caused by a complete or near-complete deficiency of the liver enzyme phenylalanine hydroxylase (PAH), which converts the amino acid phenylalanine to tyrosine, leading to the increase of blood and tissue concentration of phenylalanine to toxic levels. PKU is not life threatening but is treated through lifelong dietary management. If untreated, it can lead to severe learning disability, brain function abnormalities, behavioural and neurological problems. The non-life threatening nature of PKU has until now caused some debate on whether to licence its detection by preimplantation genetic diagnosis (PGD). We report the first successful live birth in the UK following single cell embryo biopsy and PGD for the detection of two different mutations in the (PAH) gene. This case highlights both an important scientific development as well as the ethical challenge in offering couples who carry PKU this new reproductive option when starting their family.

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Section: Methodsmentioning

confidence: 99%

Successful Live Birth following Preimplantation Genetic Diagnosis for Phenylketonuria in Day 3 Embryos by Specific Mutation Analysis and Elective Single Embryo Transfer

et al. 2012

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“…Affected embryos only have one allele that amplifies by PCR (Kakourou et al 2008). If the couple are not informative for the triplet repeat (have the same number of triplet repeats for their normal alleles) and are also not informative for linked markers, then triple-primed PCR can be used to amplify the expanded repeat to distinguish embryos with an expansion from those that are transferable (Kakourou et al 2010). Overall, the use of fluorescence has reduced the problems of contamination and time taken for the genetic analysis to be performed.…”

Section: Molecular Diagnosismentioning

confidence: 99%

Preimplantation genetic diagnosis: State of the ART 2011

2011

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For the last 20 years, preimplantation genetic diagnosis (PGD) has been mostly performed on cleavage stage embryos after the biopsy of 1-2 cells and PCR and FISH have been used for the diagnosis. The main indications have been single gene disorders and inherited chromosome abnormalities. Preimplantation genetic screening (PGS) for aneuploidy is a technique that has used PGD technology to examine chromosomes in embryos from couples undergoing IVF with the aim of helping select the chromosomally 'best' embryo for transfer. It has been applied to patients of advanced maternal age, repeated implantation failure, repeated miscarriages and severe male factor infertility. Recent randomised controlled trials (RCTs) have shown that PGS performed on cleavage stage embryos for a variety of indications does not improve delivery rates. At the cleavage stage, the cells biopsied from the embryo are often not representative of the rest of the embryo due to chromosomal mosaicism. There has therefore been a move towards blastocyst and polar body biopsy, depending on the indication and regulations in specific countries (in some countries, biopsy of embryos is not allowed). Blastocyst biopsy has an added advantage as vitrification of blastocysts, even post biopsy, has been shown to be a very successful method of cryopreserving embryos. However, mosaicism is also observed in blastocysts. There have been dramatic changes in the method of diagnosing small numbers of cells for PGD. Both array-comparative genomic hybridisation and single nucleotide polymorphism arrays have been introduced clinically for PGD and PGS. For PGD, the use of SNP arrays brings with it ethical concerns as a large amount of genetic information will be available from each embryo. For PGS, RCTs need to be conducted using both array-CGH and SNP arrays to determine if either will result in an increase in delivery rates.

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“…PCR utilizing flanking primers allows for amplification up to approximately 100 CTG repeats, but it is unreliable above this size; thus, amplification of alleles with very large numbers of CTG repeats (>100) continue to require Southern blot analysis (Falk, 2006;Tishkoff, 1998;Warner, 1996). Certain improvements in amplification of alleles with large numbers of repeats have been obtained by adding highly stable Taq DNA polymerases, and PCR-enhancing agents, such as glycerol, betaine, and 7-deaza-dGTP to the master reaction (Cheng et al, 1996;Kakourou et al, 2010;Magaña et al, 2011a;SkrzypczakZielinska et al, 2009). Currently, identification of DM1 expanded alleles is performed through fluorescent PCR and capillary electrophoresis.…”

Section: Prognosis and Diagnosis Of Dm1mentioning

confidence: 99%